Targeting the phosphoprotein enriched in diabetes/astrocytes 15 death effector domain via fragment-based and integrative structural biology approaches.

Phosphoprotein Enriched in Diabetes/Astrocytes 15 (PED/PEA-15) is a 15 kDa adaptor protein whose overexpression drives tumorigenesis and type II diabetes through dysregulated protein-protein interactions (PPIs) mediated by its Death Effector Domain (DED). Despite its therapeutic relevance, no potent small-molecule inhibitors targeting the DED have yet been identified. Here, we employed a fragment-based drug discovery approach combining 19F and 1H NMR screening of a 118-compound fluorinated library to identify F2 a fragment that binds the PED/PEA-15 DED with an estimated dissociation constant of 150 μM. NMR chemical shift perturbation analysis mapped the binding site to the α2-α3 loop, α3, and α5 helices. To better characterise the protein, Small-angle X-ray scattering of the apo protein was employed, yielding a structural model in which the C-terminal tail is consistent with a conformation that is not maximally extended. Induced fit docking and molecular dynamics simulations of the PED/PEA-15-F2 complex supported a plausible binding pose and suggested a ligand-induced destabilisation of the α3 helix propagating to the α6 helix. Correlation network analysis further suggests a hypothesis in which F2 rewires the dynamic architecture of the DED and alters allosteric communication between residues of the DED and the C-terminal tail. The identification of a ligand-accessible site on the DED supports the druggability of PED/PEA-15 and provides a refined structural framework for fragment optimisation and for testing whether engagement of this site can be translated into modulation of PED/PEA-15 PPIs with therapeutic potential in cancer and type II diabetes.